The plasminogen (Pg) system dissolves the thrombi (blood clots) that cause heart attacks and strokes. The Pg system is tightly regulated by protein-protein interactions with inhibitors, activators, substrates, etc. The cleavage of Pg to plasmin by streptokinase (SK), and other Pg activators, initiates fibrinolysis (clot dissolution) which saves the lives of heart attack patients. Recent studies have suggested that mechanistic insights into the regulation of the Pg system could further reduce the mortality from heart attacks, and improve the treatment of strokes, pulmonary embolism, etc. Because of its physiologic and therapeutic importance, our long term goal is to help elucidate the protein-protein interactions that regulate and modify the activity of the Pg system. The interactions between Pg and the indirect Pg activator SK are among the most biologically and medically important of these contacts. Studies performed in the first phase of this grant have helped to delineate the elegant interactions through which SK converts Pg (without cleavage) into the most catalytically efficient Pg activator, Insights have been made into defining the mechanisms through which: 1) SK forms a tight stable 'activator complex' with Pg (or plasmin), 2) SK non-proteolytically generates the latent active site in Pg creating a 'virgin enzyme' (Pg*), and 3) SK modifies the substrate specificity of Pg* or plasmin so that the complex can cleave Pg molecules. This continuation proposal is directed towards further dissecting the process of indirect Pg activation, in order to determine the novel mechanisms by which SK becomes a fibrin-dependent (or t-PA-like enzyme), to define whether fibrin-dependent SKs have the potential to be superior fibrinolytic agents, to understand the role of the Pg kringle domains in indirect Pg activation and, to define the intermolecular interactions that occur in the SK-Pg complex which are required for a SK-type of mechanism. In a broad scientific sense, insights into this unique process of indirect Pg activation should enlarge our understanding of how the catalytic activity and specificity of Pg system is regulated, and could suggest rational ways to alter the indirect Pg activators so as to improve their therapeutic value for patients with thrombosis.